Hippophae rhamnoides enzymolysis polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses hippophae rhamnoides enzymolysis polysaccharide as well as a preparation method and application thereof. The enzymolysis process conditions of the method are as follows: in a buffer system with the pH value of 5.0, the hippophae rhamnoides crude polysaccharide is degraded under the conditions that the addition amount of polygalacturonic acid-pectinase is 810.6 U / g, the reaction temperature is 51.3 DEG C and the enzymolysis time is 90.1 min. Structural characterization results show that the hippophae rhamnoides polysaccharide after enzymolysis is in a loose and porous form, and the specific surface area is obviously increased; and the molecular weight is greatly reduced from 1.57 * 10 Da to 4.45 * 10 Da. In-vitro activity evaluation shows that the scavenging capacity of the enzymolysis polysaccharide on DPPH free radicals is obviously superior to that of a non-enzymolysis sample; in a non-alcoholic fatty liver model constructed by HepG2 cells, the enzymolysis polysaccharide can effectively reduce the content of triglyceride and total cholesterol in the cells.

Description

Technical Field

[0001] This invention belongs to the field of chemical engineering technology, and relates to a sea buckthorn enzymatic hydrolysis polysaccharide, its preparation method and application. Background Technology

[0002] Sea buckthorn fruit polysaccharides have become a research focus due to their significant antioxidant, anti-aging, hypoglycemic, hepatoprotective, and antitumor pharmacological activities. The bioactivity of polysaccharides is closely related to their relative molecular weight: excessively high molecular weights enhance intermolecular forces and increase steric hindrance, leading to decreased solubility and thus limiting their bioavailability and application potential. Degradation treatment can effectively reduce the molecular weight and degree of polymerization of polysaccharides, improve their solubility, and thereby enhance their bioactivity. It is noteworthy that polysaccharide products obtained by different degradation methods exhibit significant differences in structure and functional properties; therefore, selecting efficient and specific degradation methods is crucial for improving the bioactivity of polysaccharides.

[0003] The main methods for degrading polysaccharides include chemical methods, physical methods, and enzymatic hydrolysis. Chemical methods suffer from poor reaction specificity, are prone to side reactions and damage the natural active structure of polysaccharides, and also involve reagent residues and environmental pollution. While physical methods avoid chemical pollution, they generally suffer from high energy consumption and poor selectivity, easily leading to non-specific degradation and affecting heat-sensitive components. In contrast, enzymatic hydrolysis, with its high specificity, can precisely cleave glycosidic bonds under mild conditions, generating products with well-defined structures and controllable molecular weight distributions. Furthermore, the process is green and leaves no harmful residues, offering significant advantages in terms of controllability and sustainability.

[0004] However, no studies have yet reported the use of enzymatic hydrolysis technology to prepare enzymatic hydrolysates with lipid-lowering and liver-protective activities from sea buckthorn polysaccharides. Therefore, developing a novel enzymatic hydrolysis method that can efficiently degrade sea buckthorn polysaccharides and prepare enzymatic hydrolysates with both lipid-lowering and liver-protective functions has become a technical challenge with significant application value that urgently needs to be overcome. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying sea buckthorn enzymatic hydrolysis of polysaccharides, thereby filling the gap in the existing technology for enzymatic hydrolysis of sea buckthorn polysaccharides.

[0006] The technical solution adopted in this invention is as follows: A method for preparing sea buckthorn enzymatic hydrolysis polysaccharide includes the following steps: 1) The crude polysaccharide of sea buckthorn prepared by water extraction and alcohol precipitation was mixed with ultrapure water, polygalacturonic acid-pectinase and citrate-disodium hydrogen phosphate buffer at pH 3-7, and enzymatically hydrolyzed at 30-70℃ for 30-150 minutes. During the enzymatic hydrolysis, the amount of pectinase added was 600-1000 U / g based on crude polysaccharide. After the enzymatic hydrolysis was completed, the mixture was centrifuged, the supernatant was collected and concentrated, and then the protein was removed by the Sevag method. Finally, the target polysaccharide precipitate was obtained by alcohol precipitation. 2) The target polysaccharide precipitate obtained in step 1) was reconstituted in water and then freeze-dried to obtain sea buckthorn enzymatically hydrolyzed crude polysaccharide; 3) The sea buckthorn enzymatically hydrolyzed crude polysaccharide was separated into polysaccharide components using an ion exchange column. The polysaccharide components were collected by elution and then subjected to dialysis, concentration, and freeze-drying in sequence to obtain sea buckthorn enzymatically hydrolyzed polysaccharide.

[0007] Furthermore, in step 1), the preparation of crude polysaccharide from sea buckthorn by water extraction and alcohol precipitation involves first extracting the polysaccharide from the sea buckthorn fruit powder with hot water, then mixing the extract with 1-3 times the volume of anhydrous ethanol or 95% ethanol solution, and finally letting it stand at 0-10 ℃ for 12-20 h.

[0008] Furthermore, in step 1), each 1g of sea buckthorn crude polysaccharide is mixed with 10-30mL of ultrapure water.

[0009] Furthermore, in step 3), the molecular weight cutoff used for dialysis is 100-300.

[0010] Further, in step 3), the crude polysaccharide from sea buckthorn is dissolved in ultrapure water and then loaded onto an ion exchange column. The packing material of the ion exchange column is anion exchange resin, and the eluent is water and 0.1-0.5 mol / L sodium chloride solution. The elution flow rate is 1-9 mL / min. The elution process is as follows: elution is performed sequentially with water, 0.1 mol / L sodium chloride solution, 0.2 mol / L sodium chloride solution, 0.3 mol / L sodium chloride solution, 0.4 mol / L sodium chloride solution, and 0.5 mol / L sodium chloride solution.

[0011] Seabuckthorn enzymatic hydrolysate prepared by any of the above methods.

[0012] The above-mentioned application of sea buckthorn enzymatic hydrolysate in the preparation of drugs and health products with lipid-lowering and liver-protecting effects.

[0013] The beneficial effects of this invention are reflected in: This invention uses crude sea buckthorn polysaccharide as raw material. By optimizing the amount of polygalacturonic acid-pectinase added, the enzymatic hydrolysis temperature, the enzymatic hydrolysis time, and the pH of the enzymatic hydrolysis buffer, combined with protein removal, alcohol precipitation, and ion exchange chromatography of the enzymatic hydrolysis extract, the extracted sea buckthorn enzymatic hydrolysate can scavenge DPPH free radicals and significantly reduce the content of intracellular triglycerides and total cholesterol. Therefore, it can be used in drugs and health products with lipid-lowering and liver-protecting effects.

[0014] This invention uses a certain amount of polygalacturonic acid-pectinase to fully hydrolyze polysaccharides under specific pH buffer, hydrolysis temperature, and hydrolysis time, thereby improving hydrolysis efficiency (reducing sugar content).

[0015] The enzymatic hydrolysis process used in this invention is mild and has a short hydrolysis time. While preserving the bioactivity of sea buckthorn enzymatic hydrolysate to the greatest extent, it also effectively avoids the problem of other active ingredients in crude sea buckthorn polysaccharides with lipid-lowering and antioxidant effects being destroyed due to prolonged exposure to high temperature and acidic / alkaline conditions.

[0016] This invention uses fiber column chromatography to separate and purify sea buckthorn enzymatic hydrolysate. Different concentrations of sodium chloride solution are used for elution in sequence, resulting in good elution and separation effect, thereby obtaining sea buckthorn enzymatic hydrolysate components with higher lipid-lowering activity. Attached Figure Description

[0017] Figure 1 This is a schematic diagram showing the changes in reducing sugar content during the enzymatic hydrolysis of sea buckthorn in this embodiment of the invention, depending on the amount of polygalacturonic acid-pectinase added (a), hydrolysis time (b), hydrolysis temperature (c), and hydrolysis pH (d).

[0018] Figure 2 The response surface methodology diagram of the sea buckthorn enzymatic hydrolysis process in this embodiment of the invention.

[0019] Figure 3 The diagram of the artificial network genetic algorithm in the seabuckthorn enzymatic hydrolysis process in this embodiment of the invention.

[0020] Figure 4 The image shows a scanning electron microscope image of sea buckthorn polysaccharide and sea buckthorn enzymatic hydrolysate in an embodiment of the present invention.

[0021] Figure 5 The infrared spectra of sea buckthorn polysaccharide and sea buckthorn enzymatic hydrolysate in the embodiments of the present invention are shown.

[0022] Figure 6 This is a molecular weight distribution diagram of sea buckthorn polysaccharide and sea buckthorn enzymatic hydrolysate in the embodiments of the present invention.

[0023] Figure 7 The image shows the elution curves of sea buckthorn enzymatic hydrolysis of polysaccharides and elution (salt washing) with sodium chloride solutions of different concentrations, as shown in the embodiments of the present invention.

[0024] Figure 8 This is a comparative diagram of the antioxidant activity of sea buckthorn polysaccharide and sea buckthorn enzymatic hydrolysate in the embodiments of the present invention.

[0025] Figure 9 This figure shows the effect of sea buckthorn polysaccharide and sea buckthorn enzymatic hydrolysate on the triglyceride content in HepG2 cells in an embodiment of the present invention.

[0026] Figure 10 This figure shows the effect of sea buckthorn polysaccharide and sea buckthorn enzymatic hydrolysate on the total cholesterol content in HepG2 cells in an embodiment of the present invention. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0028] To address the issues of large molecular weight, relatively low bioactivity, and low purity of sea buckthorn polysaccharides obtained through enzymatic hydrolysis, this invention proposes a method for preparing enzymatically hydrolyzed sea buckthorn polysaccharides and their applications. (I) A method for preparing sea buckthorn enzymatic hydrolysate polysaccharide, comprising the following steps: 1) The crude polysaccharide of sea buckthorn prepared by water extraction and alcohol precipitation was mixed with ultrapure water, polygalacturonic acid-pectinase and citrate-disodium hydrogen phosphate buffer at pH 3-7, and enzymatically hydrolyzed at 30-70℃ for 30-150 minutes. During the enzymatic hydrolysis, the amount of pectinase added was 600-1000 U / g based on crude polysaccharide. After the enzymatic hydrolysis was completed, the mixture was centrifuged, the supernatant was collected and concentrated, and then the protein was removed by the Sevag method. Finally, the target polysaccharide precipitate was obtained by alcohol precipitation. 2) The target polysaccharide precipitate obtained in step 1) was reconstituted in water and then freeze-dried to obtain sea buckthorn enzymatically hydrolyzed crude polysaccharide; 3) The sea buckthorn enzymatically hydrolyzed crude polysaccharide was separated into polysaccharide components using an ion exchange column. The polysaccharide components were collected by elution and then subjected to dialysis, concentration, and freeze-drying in sequence to obtain sea buckthorn enzymatically hydrolyzed polysaccharide.

[0029] Preparation of citrate-disodium hydrogen phosphate buffer (pH 3.0-7.0): Dissolve 7.16 g of disodium hydrogen phosphate and 10 g of citric acid in 100 mL of ultrapure water. Adjust the pH to the required level by adding 0.2 mol / L disodium hydrogen phosphate and 0.1 mol / L citric acid.

[0030] Optimization of process parameters for enzymatic hydrolysis of polysaccharides from sea buckthorn, such as Figure 1 As shown: In (a), the enzymatic hydrolysis time was fixed at 60 min, the enzymatic hydrolysis temperature at 40 °C, and the enzymatic hydrolysis pH at 4.0.

[0031] In (b), the amount of fixed enzyme added was 800 U / g, the enzymatic hydrolysis temperature was 40℃, and the enzymatic hydrolysis pH was 4.0.

[0032] In (c), the amount of fixed enzyme added was 800 U / g, the enzymatic hydrolysis time was 90 min, and the enzymatic hydrolysis pH was 4.0.

[0033] In (d), the amount of fixed enzyme added was 800 U / g, the enzymatic hydrolysis time was 90 min, and the enzymatic hydrolysis temperature was 50℃.

[0034] like Figure 2 As shown, based on the single-factor experiments, with the amount of reducing sugar produced as the indicator, the enzyme addition amount (700, 800 and 900 U / g), enzymatic hydrolysis time (60, 90 and 120 min), enzymatic hydrolysis temperature (40, 50 and 60 ℃) and enzymatic hydrolysis pH (4, 5 and 6) were selected. According to the Box-Behnken design principle, a four-factor, three-level response surface analysis experiment was conducted using Design Expert 13.0.6 software.

[0035] like Figure 3 As shown, an artificial neural network-genetic algorithm model was constructed to optimize the enzymatic hydrolysis process of sea buckthorn polysaccharides. The model structure consists of one input layer, two hidden layers, and one output layer. The input layer receives four feature parameters: enzyme addition amount, hydrolysis time, hydrolysis temperature, and hydrolysis pH; the two hidden layers contain 12 and 18 neurons, respectively; the output layer has one neuron, which outputs a comprehensive evaluation value with reducing sugar content as the main indicator. The optimal solution for the enzymatic hydrolysis process of sea buckthorn polysaccharides is predicted by this model.

[0036] Example of preparation of sea buckthorn enzymatic hydrolysis polysaccharide The enzymatic hydrolysis of crude sea buckthorn polysaccharide was completed by selecting process parameters with high reducing sugar content, and salt washing experiments of polysaccharide were carried out using sodium chloride solutions of different concentrations in fiber column chromatography.

[0037] (1) After crushing the sea buckthorn fruit, crude sea buckthorn polysaccharide was obtained by water extraction and alcohol precipitation. Add 30 mL of deionized water and polygalacturonic acid-pectinase to 1 g of crude sea buckthorn polysaccharide according to the material-to-liquid ratio, then add 4 mL of citrate-disodium hydrogen phosphate buffer solution. Place the mixed enzymatic hydrolysis system in a water bath and perform enzymatic hydrolysis at pH [missing value]. 5. Enzymatic hydrolysis was performed at 50℃ for 90 min. After hydrolysis, the enzyme was inactivated by boiling water bath for 10 min to obtain the extract. The extract was centrifuged using a high-speed centrifuge to remove insoluble matter. Then, it was concentrated under reduced pressure using a rotary evaporator (50℃ water temperature) to reduce the water content in the extract. Sevage reagent (chloroform: n-butanol = 4:1, v / v) was added to the concentrate, which was shaken and centrifuged to remove protein. Then, four volumes of anhydrous ethanol were added to the supernatant obtained by centrifugation for alcohol precipitation (standing overnight at 4℃). The alcohol precipitate was concentrated under reduced pressure using a rotary evaporator (50℃ water temperature) to remove ethanol from the solution. The precipitate was collected by centrifugation at low temperature (4℃). (2) Take the precipitate obtained by alcohol precipitation in step (1), redissolve it in deionized water and freeze dry it to obtain sea buckthorn enzymatic hydrolysis crude polysaccharide with a yield of 5.89%. The polysaccharide content was determined to be 56.75% by phenol-sulfuric acid method. (3) Take 0.50 g of the crude polysaccharide from step (2), dissolve it in 50 mL of deionized water, and load it onto a DEAE-52 cellulose column for separation. Elute sequentially with water, 0.1 mol / L sodium chloride solution, 0.2 mol / L sodium chloride solution, 0.3 mol / L sodium chloride solution, 0.4 mol / L sodium chloride solution, and 0.5 mol / L sodium chloride solution at a flow rate of 8 mL / min. Detect the absorbance of the polysaccharide in the eluent using the phenol-sulfuric acid method. Collect the eluent using an automatic collector, collecting one tube of eluent every 8 min. Collect the eluent from tubes 1-200 eluted with deionized water, tubes 201-300 eluted with 0.1 mol / L sodium chloride solution, tubes 301-400 eluted with 0.2 mol / L sodium chloride solution, tubes 401-500 eluted with 0.3 mol / L sodium chloride solution, and 0.4 mol / L sodium chloride solution. The eluents from tubes 401-500 eluted with 0.5 mol / L sodium chloride solution and tubes 501-600 eluted with 0.5 mol / L sodium chloride solution were placed in dialysis bags with a molecular weight cutoff of 300 and dialyzed in deionized water for 48 h. The eluents were then concentrated under reduced pressure using a rotary evaporator (at a water temperature of 50 °C) to reduce the water content. Subsequently, the eluents were freeze-dried at -50 °C for 48 h to obtain sea buckthorn enzymatic hydrolysate. The results are as follows Figure 7As shown, the sea buckthorn polysaccharide was eluted sequentially with deionized water, 0.1 mol / L sodium chloride solution, 0.2 mol / L sodium chloride solution, 0.3 mol / L sodium chloride solution, 0.4 mol / L sodium chloride solution, and 0.5 mol / L sodium chloride solution. The final yields of sea buckthorn enzymatic hydrolysis polysaccharide (the ratio of the mass of sea buckthorn enzymatic hydrolysis polysaccharide to the mass of the sample polysaccharide) were 4.4%, 10.84%, 26.00%, 19.04%, and 9.60%, respectively.

[0038] (II) Morphological characterization of sea buckthorn enzymatic hydrolysate polysaccharides 1.1 Take appropriate amounts of dried sea buckthorn polysaccharide samples and dried sea buckthorn enzymatically hydrolyzed polysaccharide samples, adhere them to a sample stage with copper tape, and place the sample stage in an ion sputtering instrument to deposit a layer of conductive gold powder; then place it under an S-3400N scanning electron microscope for observation. Working conditions: accelerating voltage 15 kV, magnification 100x. Perform corresponding sharpness adjustments until an ideal field of view is obtained, and select an appropriate field of view to take pictures and record. Each sample is photographed three times to eliminate sample interference and systematic errors. The results are as follows: Figure 4 As shown.

[0039] 1.2 Take appropriate amounts of dried sea buckthorn polysaccharide samples and dried sea buckthorn enzymatically hydrolyzed polysaccharide samples, add appropriate amounts of potassium bromide, compress into tablets, and measure the 4000-500 cm⁻¹ using an infrared absorption spectrometer. -1 The infrared absorption spectrum within the range, the results are as follows Figure 5 As shown.

[0040] 1.3 The molecular weight of dried sea buckthorn polysaccharide samples and dried sea buckthorn enzymatic hydrolysate samples was determined by gel permeation chromatography. The analysis was performed on an Agilent GPC / SEC high-performance liquid chromatography system equipped with a THU-H2O column and a differential refractive index detector. Aqueous solution was used as the mobile phase at a flow rate of 1.0 mL / min. -1 The injection volume was 50 μL, and the results were as follows: Figure 6 As shown.

[0041] (III) In vitro antioxidant experiment of sea buckthorn enzymatic hydrolysis polysaccharide 0.2 mol / L DPPH anhydrous ethanol solution was added to both sea buckthorn polysaccharide and sea buckthorn enzymatic hydrolysate solutions. After mixing, the solutions were incubated in a water bath at 25 ℃ in the dark for 30 min, and the absorbance was measured at 517 nm. A vitamin C solution of the same concentration as the polysaccharide solution was used as a positive control. The results are as follows: Figure 8 As shown.

[0042] (iv) In vitro lipid-lowering experiment of sea buckthorn enzymatic hydrolysis polysaccharide In a non-alcoholic fatty liver model based on HepG2 cells, the effect of sea buckthorn enzymatic hydrolysis on the clearance of intracellular triglycerides and total cholesterol was demonstrated.

[0043] Experimental groups: normal group, model group (250 mmol / L OA + 500 mmol / L PA), positive control group (simvastatin), sea buckthorn polysaccharide group (SBP) and enzymatic hydrolysis of sea buckthorn polysaccharide group (ESBP).

[0044] (1) Triglyceride clearance rate HepG2 cells were cultured in complete medium (DMEM, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution) at 37°C and 5% CO2. Cells were passaged routinely every 2-3 days, with cells in the logarithmic growth phase selected at a density of 5 × 10⁶ cells per well. 5 Individual samples were inoculated into 12-well plates, and different concentrations of the drug were added for 24 hours of intervention. Subsequent procedures were performed according to the instructions provided by the reagent kit manufacturer. The results are as follows: Figure 9 As shown.

[0045] (2) Total cholesterol clearance rate HepG2 cells were cultured in complete medium (DMEM, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution) at 37°C and 5% CO2. Cells were passaged routinely every 2-3 days, with cells in the logarithmic growth phase selected at a density of 5 × 10⁶ cells per well. 5 Individual samples were inoculated into 12-well plates, and different concentrations of the drug were added for 24 hours of intervention. Subsequent procedures were performed according to the instructions provided by the reagent kit manufacturer. The results are as follows: Figure 10 As shown.

[0046] In summary, this invention employs optimized enzymatic hydrolysis conditions in the enzymatic hydrolysis of sea buckthorn polysaccharides. The resulting hydrolysate is centrifuged, concentrated, deproteinized, precipitated with alcohol, reconstituted, and freeze-dried to obtain crude sea buckthorn enzymatically hydrolyzed polysaccharides. The crude polysaccharides are then separated and purified using fiber column chromatography. The eluent is dialyzed, concentrated, and freeze-dried to obtain the final sea buckthorn enzymatically hydrolyzed polysaccharide. The enzymatic hydrolysis process of this invention is mild and has a short hydrolysis time. While maximizing the preservation of the bioactivity of the sea buckthorn enzymatically hydrolyzed polysaccharides, it also effectively avoids the problem of damage to other active components with lipid-lowering and antioxidant effects caused by prolonged exposure to high-temperature and acidic / alkaline conditions. This provides a new avenue for the further development of sea buckthorn polysaccharides.

Claims

1. A method for preparing a seabuckthorn enzymatic polysaccharide, characterized by: The method comprises the following steps: 1) mixing the sea-buckthorn crude polysaccharide prepared by water extraction and alcohol precipitation with ultrapure water, polygalacturonase-pectinase and a citric acid-disodium hydrogen phosphate buffer solution with a pH of 3-7, and carrying out enzymatic hydrolysis at 30-70°C for 30-150 minutes, wherein the addition amount of pectinase is 600-1000 U / g of the crude polysaccharide during the enzymatic hydrolysis, and after the enzymatic hydrolysis, the mixture is centrifuged, the supernatant is collected and concentrated, then the protein in the supernatant is removed by the Sevag method, and finally, the target polysaccharide precipitate is obtained by alcohol precipitation; 2) freeze-drying the target polysaccharide precipitate obtained in step 1) after being redissolved in water to obtain sea-buckthorn enzymatic crude polysaccharide; 3) separating the sea-buckthorn enzymatic crude polysaccharide by ion exchange column to obtain polysaccharide components, collecting the polysaccharide components by elution, and then carrying out dialysis, concentration and freeze-drying in sequence to obtain sea-buckthorn enzymatic polysaccharide.

2. The preparation method of sea buckthorn enzymatic polysaccharide according to claim 1, characterized in that: In step 1), the sea-buckthorn crude polysaccharide prepared by water extraction and alcohol precipitation is first extracted from sea-buckthorn fruit powder with hot water, then the extraction liquid is mixed with 1-3 times the volume of anhydrous ethanol or a 95% ethanol solution, and finally, the mixture is placed at 0-10°C for 12-20 hours.

3. The method for preparing sea buckthorn enzymatic polysaccharide according to claim 2, characterized in that: In step 1), 10-30 mL of ultrapure water is mixed with 1 g of sea-buckthorn crude polysaccharide.

4. The method for preparing sea buckthorn enzymatic polysaccharide according to claim 3, characterized in that: In step 3), the molecular weight cut-off for dialysis is 100-300.

5. The method for preparing sea buckthorn enzymatic polysaccharide according to claim 4, characterized in that: In step 3), the sea-buckthorn enzymatic crude polysaccharide is dissolved in ultrapure water and then loaded onto an ion exchange column, the filler of the ion exchange column is an anion exchange resin, the eluent is water and a 0.1-0.5 mol / L sodium chloride solution, and the elution flow rate is 1-9 mL / min; the elution process comprises the following steps: sequentially eluting with water, a 0.1 mol / L sodium chloride solution, a 0.2 mol / L sodium chloride solution, a 0.3 mol / L sodium chloride solution, a 0.4 mol / L sodium chloride solution and a 0.5 mol / L sodium chloride solution.

6. The sea-buckthorn enzymatic polysaccharide prepared by the preparation method of sea-buckthorn enzymatic polysaccharide according to any one of claims 1-5.

7. The sea-buckthorn enzymatic polysaccharide for use in the preparation of a drug or health care product with lipid-lowering and liver-protecting effects according to claim 6.

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